Little is known about the characteristics of cystine transport and expression of cystine transporters in heart. Such knowledge is likely to be important because the cystine-glutamate exchanger (xCT) has been suggested to be a rate-limiting factor in glutathione synthesis (Sato et al. 1998), and glutathione plays a major role in protecting heart cells against oxidative stress (Dhalla et al. 2000). The aims of this study were to investigate cystine uptake in cardiac sarcolemmal vesicles under normal conditions and during ischaemia.

Male Wistar rats were humanely killed by cervical dislocation. The hearts were dissected and were either used directly for sarcolemmal vesicle preparation or perfused as Langendorrf preparations before sarcolemmal vesicle preparation. Langendorrf hearts were perfused at 10 ml min^-1 with aerated Krebs solution at 37 °C (Javadov et al. 2000) for 45 min with (ischaemic) or without (control) 30 min global normothermic ischaemia. Homogenisation and differential centrifugation was used to prepare the vesicles as described previously with the purity of the vesicle preparation assayed by marker enzyme activity (King et al. 2001). L-[¹⁴C]Cystine uptake into sarcolemmal vesicles was measured by rapid filtration at room temperature (King et al. 2001).

There was no significant difference in the enrichment of Na⁺,K⁺-ATPase activity in vesicles from control compared to ischaemic hearts (6.67 ± 1.15-fold control vs. 8.53 ± 1.01-fold ischaemic, n = 3, means ± S.E.M.). L-[¹⁴C]Cystine uptake into sarcolemmal vesicles was not sodium dependent but was affected by ischaemia and by pH. The initial rate of 0.03 mM L-[¹⁴C]cystine uptake in vesicles from ischaemic hearts was 66.18 ± 2.91 pmol (mg protein) s^-1, which was significantly greater than the 29.86 ± 10.98 pmol (mg protein) s^-1 measured in vesicles from control hearts (P < 0.05, Student’s unpaired t test, n = 3, means ± S.E.M.). When the pH was decreased, the initial rate of 0.1 mM L-[¹⁴C]cystine uptake increased from 10.58 ± 6.86 pmol (mg protein) s^-1 at pH 7.4 to 104.44 ± 39.73 pmol (mg protein) s^-1 at pH 6.2 (P < 0.05, t test, n = 3 ± S.D.) in vesicles from control hearts.

This work suggests that cystine uptake into rat cardiac sarcolemmal vesicles is stimulated by low pH and ischaemia.

This work was supported by the British Heart Foundation.